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1.
Plant Cell Physiol ; 60(8): 1633-1645, 2019 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-31292642

RESUMO

Plants respond to a rise in ambient temperature by increasing the growth of petioles and hypocotyls. In this work, we show that Arabidopsis thaliana class I TEOSINTE BRANCHED 1, CYCLOIDEA, PCF (TCP) transcription factors TCP14 and TCP15 are required for optimal petiole and hypocotyl elongation under high ambient temperature. These TCPs influence the levels of the DELLA protein RGA and the expression of growth-related genes, which are induced in response to an increase in temperature. However, the class I TCPs are not required for the induction of the auxin biosynthesis gene YUCCA8 or for auxin-dependent gene expression responses. TCP15 directly targets the gibberellin biosynthesis gene GA20ox1 and the growth regulatory genes HBI1 and PRE6. Several of the genes regulated by TCP15 are also targets of the growth regulator PIF4 and show an enrichment of PIF4- and TCP-binding motifs in their promoters. PIF4 binding to GA20ox1 and HBI1 is enhanced in the presence of the TCPs, indicating that TCP14 and TCP15 directly participate in the induction of genes involved in gibberellin biosynthesis and cell expansion by high temperature functionally interacting with PIF4. In addition, overexpression of HBI1 rescues the growth defects of tcp14 tcp15 double mutants, suggesting that this gene is a major outcome of regulation by both class I TCPs during thermomorphogenesis.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Giberelinas/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Regulação da Expressão Gênica de Plantas , Oxigenases de Função Mista/genética , Oxigenases de Função Mista/metabolismo , Complexo de Endopeptidases do Proteassoma/genética , Temperatura Ambiente , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
2.
Parasit Vectors ; 12(1): 337, 2019 Jul 08.
Artigo em Inglês | MEDLINE | ID: mdl-31287014

RESUMO

BACKGROUND: Aedes aegypti were found developing in the water in open public drains (drain-water, DW) in Jaffna city in northern Sri Lanka, a location where the arboviral diseases dengue and chikungunya are endemic. METHODS: Susceptibilities to the common insecticides dichlorodiphenyltrichloroethane (DDT), malathion, propoxur, permethrin and deltamethrin and activities of the insecticide-detoxifying enzymes carboxylesterase (EST), glutathione S-transferase (GST) and monooxygenase (MO) were compared in adult Ae. aegypti developing in DW and fresh water (FW). RESULTS: DW Ae. aegypti were resistant to the pyrethroids deltamethrin and permethrin, while FW Ae. aegypti were susceptible to deltamethrin but possibly resistant to permethrin. Both DW and FW Ae. aegypti were resistant to DDT, malathion and propoxur. Greater pyrethroid resistance in DW Ae. aegypti was consistent with higher GST and MO activities. CONCLUSIONS: The results demonstrate the potential for insecticide resistance developing in Ae. aegypti adapted to DW. Urbanization in arboviral disease-endemic countries is characterized by a proliferation of open water drains and therefore the findings identify a potential new challenge to global health.


Assuntos
Aedes/enzimologia , Arbovirus/fisiologia , Resistência a Inseticidas , Mosquitos Vetores/enzimologia , Águas Residuárias/parasitologia , Aedes/efeitos dos fármacos , Aedes/virologia , Animais , Carboxilesterase/metabolismo , DDT/farmacologia , Feminino , Saúde Global , Glutationa Transferase/metabolismo , Humanos , Inseticidas/farmacologia , Malation/farmacologia , Masculino , Camundongos , Oxigenases de Função Mista/metabolismo , Mosquitos Vetores/efeitos dos fármacos , Mosquitos Vetores/virologia , Nitrilos/farmacologia , Permetrina/farmacologia , Propoxur/farmacologia , Piretrinas/farmacologia
3.
Life Sci ; 232: 116601, 2019 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-31252000

RESUMO

AIMS: Tet1, Tet2, and interleukin-6 (IL-6) have been linked to atherosclerosis. Whether Tet3 has a relationship with atherosclerosis and IL-6 was unknown. This study aims to determine the link between Tet3 and IL-6, and the role of Tet3 in prenatal hypoxia-induced atherosclerosis in offspring rats. MAIN METHODS: Pregnant rats were divided into hypoxia and control group. Their male offspring were tested at 20 months old. Hematoxylin-eosin staining and transmission electron microscopic staining were used. Gene mRNA and protein levels were measured with q-PCR or Western blotting. Cell viability and migration was tested with MTT or cell scratch assay. 5-hmC and 5-mC expression were obtained by qGlucMS-PCR; 5-hmC and 5-mC activity were obtained by dot blotting. KEY FINDINGS: Chronic prenatal hypoxia increased Tet3 and IL-6 expression, and decreased Tet3 activity in offspring rats. GlucMS-qPCR showed the percentage of 5-hmC was significantly up-regulated in the promoter of IL-6 in both the rats and cells. Moreover, 5-hmC percentage also was increased in the A7r5 cells transfected with Tet3. Furthermore, Tet3 promoted proliferation and migration of A7r5 cells. However, Tet3 was not sensitive to acute hypoxia, while influenced by HIF-1α DNA element. SIGNIFICANCE: Tet3 enhanced IL-6 expression though up-regulating 5-hmC percentage in the IL-6 promoter.


Assuntos
Aterosclerose/metabolismo , Desoxicitidina/análogos & derivados , Dioxigenases/metabolismo , Hipóxia/metabolismo , Interleucina-6/biossíntese , Animais , Aterosclerose/genética , Aterosclerose/patologia , Movimento Celular/fisiologia , Sobrevivência Celular/fisiologia , Desoxicitidina/metabolismo , Epigênese Genética , Feminino , Hipóxia/genética , Hipóxia/patologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Interleucina-6/genética , Interleucina-6/metabolismo , Masculino , Oxigenases de Função Mista/metabolismo , Gravidez , Complicações na Gravidez/metabolismo , Complicações na Gravidez/patologia , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Ativação Transcricional , Regulação para Cima
4.
Food Chem ; 295: 82-93, 2019 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-31174813

RESUMO

The aim of the study was to investigate the occurrence and formation of peroxygenase catalysed non-volatile oxidised fatty acids (NVOFAs), especially epoxy and hydroxy fatty acids, which potentially provide off-flavours in oat. A method based on extraction of NVOFAs using accelerated solvent extraction and analysis by UHPLC-ELSD/MS was developed. Hydroxy and epoxy fatty acids from oleic and linoleic acids were present as major NVOFAs in non-heat treated (NHT) oat products, and the contents increased markedly during storage. In a controlled storage experiment, NVOFA contents in NHT oat flours increased to 1700-2000 µg/g, whereas in heat-treated samples, only to 200-400 µg/g. Epoxy fatty acids seemed to be the first products that occurred, followed by hydroxy fatty acids and minor NVOFAs. The formation of NVOFAs was related to lipase catalysed lipid hydrolysis and the formation of volatile lipid oxidation products. Inactivation of lipid degrading enzymes is crucial to producing stable oat products.


Assuntos
Avena/metabolismo , Ácidos Graxos/análise , Biocatálise , Cromatografia Líquida de Alta Pressão , Ácidos Graxos/isolamento & purificação , Farinha/análise , Ácidos Linoleicos , Peroxidação de Lipídeos , Espectrometria de Massas , Oxigenases de Função Mista/metabolismo , Extração em Fase Sólida
5.
Carbohydr Res ; 478: 46-53, 2019 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-31054382

RESUMO

A novel Lytic Polysaccharide Monooxygenase (LPMO) family AA9 (PMO9A_MALCI) protein from thermophilic fungus Malbranchea cinnamomea was cloned and expressed in Pichia pastoris. The expressed protein was purified to homogeneity using ion exchange and hydrophobic interaction chromatography. SDS-PAGE analysis showed PMO9A_MALCI to be ~27 kDa protein. High performance anion exchange chromatography and mass spectrometry confirmed that purified protein was active against an array of cellulosic (avicel, carboxy methyl cellulose) and hemicellulosic (birch wood xylan, wheat arabinoxylan and rye arabinoxylan) substrates, releasing both oxidized and unoxidized cello-oligosaccharide and xylo-oligosaccharide products respectively. Presence of double oxidized products during mass spectrometric analysis as well as in-silico analysis confirmed that the expressed protein belongs to Type 3 LPMO family. Molecular dynamic simulations further confirmed the sharing of common amino acid residues conserved for catalysis of both cellulosic and hemicellulosic substrates which further indicates that both substrates are equally preferred. Enzymatic cocktails constituted by replacing a part of commercial cellulase CellicCTec2 with PMO9A_MALCI (9:1/8:2) led to synergistic improvement in saccharification of acid and alkali pretreated biomass. This is the first report on heterologous expression of LPMO from M. cinnamomea, exhibiting catalysis of cellulose and pure xylan.


Assuntos
Ascomicetos/enzimologia , Oxigenases de Função Mista/metabolismo , Polissacarídeos/metabolismo , Biocatálise , Configuração de Carboidratos , Oxigenases de Função Mista/química , Simulação de Acoplamento Molecular , Polissacarídeos/química
6.
Plant Sci ; 283: 301-310, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31128700

RESUMO

Mandrakes (Mandragora spp., Solanaceae) are known to contain tropane alkaloids and have been used since antiquity in traditional medicine. Tropane alkaloids such as scopolamine and hyoscyamine are used in modern medicine to treat pain, motion sickness, as eye pupil dilators and antidotes against organo-phosphate poisoning. Hyoscyamine is converted to 6ß-hydroxyhyoscyamine (anisodamine) and scopolamine by hyoscyamine 6ß-hydroxylase (H6H), a 2-oxoglutarate dependent dioxygenase. We describe here a marked chemo-diversity in the tropane alkaloid content in Mandragora spp. M. officinarum and M. turcomanica lack anisodamine and scopolamine but display up to 10 fold higher hyoscyamine levels as compared with M. autumnalis. Transcriptomic analyses revealed that H6H is highly conserved among scopolamine-producing Solanaceae. MoH6H present in M. officinarum differs in several amino acid residues including a homozygotic mutation in the substrate binding region of the protein and its prevalence among accessions was confirmed by Cleaved-Amplified-Polymorphic-Sequence analyses. Functional expression revealed that MaH6H, a gene isolated from M. autumnalis encodes an active H6H enzyme while the MoH6H sequence isolated from M. officinarum was functionally inactive. A single G to T mutation in nucleotide 663 of MoH6H is associated with the lack of anisodamine and scopolamine in M. officinalis.


Assuntos
Alcaloides/metabolismo , Mandragora/metabolismo , Oxigenases de Função Mista/metabolismo , Perfilação da Expressão Gênica , Genes de Plantas/genética , Mandragora/genética , Oxigenases de Função Mista/genética , Escopolamina/metabolismo , Análise de Sequência de DNA , Alcaloides de Solanáceas/metabolismo
7.
Chemosphere ; 231: 173-183, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31129398

RESUMO

Our understanding of the tetrahydrofuran (THF) degradation in complex environment is limited. The majority of THF degrading genes reported are group V soluble diiron monooxygenases and share greater than 95% homology with one another. In this study, we used sole-carbon-source incubation combined with high-throughput metagenomic sequencing to investigate this contaminant's degradation in environmental samples. We identified as-yet-uncultivated microbe from the genera Pseudonocardia and fungi Scedosporium sp. (Scedosporium sp. was successfully isolated) as THF degraders as containing THF degradation genes, while microbes from the genera Bordetella, Pandoraea and Rhodanobacter functioned as main cooperators by utilizing acidic intermediates and providing anti-acid mechanisms. Furthermore, a 9387-bp THF degradation cluster designated thmX from the as-yet-uncultivated Pseudonocardia (with 6 main ORFs and with 79-93% amino acid sequence identity with previously reported clusters) was discovered. We also found a THF-degrading related cytochrome P450 monooxygenase from the genus Scedosporium and predicted its cognate reductase for the first time. All the genes and clusters mentioned above were successfully amplified from samples and cloned into the suitable expression vectors. This study will provide novel insights for understanding of THF degradation mechanisms under acid stress conditions and mining new THF degradation genes.


Assuntos
Furanos/química , Metagenoma/genética , Actinomycetales , Furanos/análise , Furanos/metabolismo , Metagenômica , Microbiota , Oxigenases de Função Mista/genética , Oxigenases de Função Mista/metabolismo , Fases de Leitura Aberta
8.
Nat Commun ; 10(1): 1994, 2019 04 30.
Artigo em Inglês | MEDLINE | ID: mdl-31040279

RESUMO

Lignin biosynthesis is evolutionarily conserved among higher plants and features a critical 3-hydroxylation reaction involving phenolic esters. However, increasing evidence questions the involvement of a single pathway to lignin formation in vascular plants. Here we describe an enzyme catalyzing the direct 3-hydroxylation of 4-coumarate to caffeate in lignin biosynthesis as a bifunctional peroxidase that oxidizes both ascorbate and 4-coumarate at comparable rates. A combination of biochemical and genetic evidence in the model plants Brachypodium distachyon and Arabidopsis thaliana supports a role for this coumarate 3-hydroxylase (C3H) in the early steps of lignin biosynthesis. The subsequent efficient O-methylation of caffeate to ferulate in grasses is substantiated by in vivo biochemical assays. Our results identify C3H as the only non-membrane bound hydroxylase in the lignin pathway and revise the currently accepted models of lignin biosynthesis, suggesting new gene targets to improve forage and bioenergy crops.


Assuntos
Citosol/enzimologia , Lignina/biossíntese , Arabidopsis/metabolismo , Ascorbato Peroxidases , Brachypodium/metabolismo , Ácidos Cafeicos/metabolismo , Ácidos Cumáricos/metabolismo , Oxigenases de Função Mista/genética , Oxigenases de Função Mista/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo
9.
BMC Plant Biol ; 19(1): 195, 2019 May 14.
Artigo em Inglês | MEDLINE | ID: mdl-31088366

RESUMO

BACKGROUND: Flavonol synthase (FLS) is the key enzyme responsible for the biosynthesis of flavonols, the most abundant flavonoids, which have diverse pharmaceutical effects. Flavonol synthase has been previously found in other species, but not yet in Ornithogalum caudatum. RESULTS: The transcriptome-wide mining and functional characterisation of a flavonol synthase gene family from O. caudatum were reported. Specifically, a small FLS gene family harbouring two members, OcFLS1 and OcFLS2, was isolated from O. caudatum based on transcriptome-wide mining. Phylogenetic analysis suggested that the two proteins showed the closest relationship with FLS proteins. In vitro enzymatic assays indicated OcFLS1 and OcFLS2 were flavonol synthases, catalysing the conversion of dihydroflavonols to flavonols in an iron-dependent fashion. In addition, the two proteins were found to display flavanone 3ß-hydroxylase (F3H) activity, hydroxylating flavanones to form dihydroflavonols. Unlike single F3H enzymes, the F3H activity of OcFLS1 and OcFLS2 did not absolutely require iron. However, the presence of sufficient Fe2+ was demonstrated to be conducive to successive catalysis of flavanones to flavonols. The qRT-PCR analysis demonstrated that both genes were expressed in the leaves, bulbs, and flowers, with particularly high expression in the leaves. Moreover, their expression was regulated by developmental and environmental conditions. CONCLUSIONS: OcFLS1 and OcFLS2 from O. caudatum were demonstrated to be flavonol synthases with iron-independent flavanone 3-hydroxylase activity.


Assuntos
Oxigenases de Função Mista/metabolismo , Ornithogalum/enzimologia , Oxirredutases/metabolismo , Proteínas de Plantas/metabolismo , Eletroforese em Gel de Poliacrilamida , Flavonóis/metabolismo , Perfilação da Expressão Gênica , Genes de Plantas/genética , Genes de Plantas/fisiologia , Ferro/metabolismo , Redes e Vias Metabólicas , Ornithogalum/genética , Ornithogalum/metabolismo , Análise de Sequência de DNA , Transcriptoma
10.
Curr Microbiol ; 76(6): 698-705, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30955044

RESUMO

Group A streptococcus (GAS) is an important human pathogen whose clinical isolates differ in their ability to produce hydrogen peroxide (H2O2). H2O2 is primarily produced by the enzyme lactate oxidase (LctO), an in depth in silico research revealed that all genome-sequenced GAS possess the required gene lctO. The importance of lctO for GAS is underlined by its highly conserved catabolite control element (cre box) as well as its perfect promotor sequence in comparison to the known consensus sequences of the Gram-positive model organism Bacillus subtilis. In this study, we provide further insight in the function and regulation of lactate oxidase by analyzing a large group of clinical GAS isolates. We found that H2O2 production increased over time in the late stationary phase; after 4 days of incubation, 5.4% of the isolates showed a positive result at 37 °C, while the rate increased to 16.4% at 20 °C. This correlation between H2O2 production and low temperatures suggests additional regulatory mechanisms for lctO besides catabolite control protein A (CcpA) and indicates that lctO might play a role for GAS energy metabolism at sub-body temperatures. Furthermore, we could identify that H2O2 production was different among clinical isolates; we could correlate H2O2 production to emm-types, indicating that emm-types 6 and 75 had the highest rate of H2O2 production. The emm-type- and temperature-dependent H2O2 production of clinical GAS isolates might contribute to their different survival strategies.


Assuntos
Antígenos de Bactérias/análise , Proteínas da Membrana Bacteriana Externa/análise , Proteínas de Transporte/análise , Peróxido de Hidrogênio/metabolismo , Oxigenases de Função Mista/genética , Oxigenases de Função Mista/metabolismo , Oxidantes/metabolismo , Streptococcus pyogenes/metabolismo , Streptococcus pyogenes/efeitos da radiação , Proteínas de Bactérias/metabolismo , Metabolismo Energético , Regulação Bacteriana da Expressão Gênica , Humanos , Proteínas Repressoras/metabolismo , Infecções Estreptocócicas/microbiologia , Streptococcus pyogenes/classificação , Streptococcus pyogenes/genética , Temperatura Ambiente
11.
Microb Cell Fact ; 18(1): 74, 2019 Apr 25.
Artigo em Inglês | MEDLINE | ID: mdl-31023316

RESUMO

BACKGROUND: Production of L-tyrosine is gaining grounds as the market size of 3,4-dihydroxyphenyl-L-alanine (L-DOPA) is expected to increase due to increasing cases of Parkinson's disease a neurodegenerative disease. Attempts to overproduce L-tyrosine for conversion to L-DOPA has stemmed on the overexpressing of critical pathway enzymes, an introduction of feedback-resistant enzymes, and deregulation of transcriptional regulators. RESULTS: An E. coli BL21 (DE3) was engineered by deleting tyrR, ptsG, crr, pheA and pykF while directing carbon flow through the overexpressing of galP and glk. TktA and PpsA were also overexpressed to enhance the accumulation of E4P and PEP. Directed evolution was then applied on HpaB to optimize its activity. Three mutants, G883R, G883A, L1231M, were identified to have improved activity as compared to the wild-type hpaB showing a 3.03-, 2.9- and 2.56-fold increase in L-DOPA production respectively. The use of strain LP-8 resulted in the production of 691.24 mg/L and 25.53 g/L of L-DOPA in shake flask and 5 L bioreactor, respectively. CONCLUSION: Deletion of key enzymes to channel flux towards the shikimate pathway coupled with the overexpression of pathway enzymes enhanced the availability of L-tyrosine for L-DOPA production. Enhancing the activity of HpaB increased L-DOPA production from glucose and glycerol. This work demonstrates that increasing the availability of L-tyrosine and enhancing enzyme activity ensures maximum L-DOPA productivity.


Assuntos
Escherichia coli/metabolismo , Glucose/metabolismo , Levodopa/biossíntese , Engenharia Metabólica , Tirosina/metabolismo , Reatores Biológicos , Escherichia coli/genética , Glicerol/metabolismo , Oxigenases de Função Mista/metabolismo , Ácido Chiquímico/metabolismo , Tirosina/genética
12.
Bioelectrochemistry ; 128: 66-73, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-30928867

RESUMO

Different carbon-based nanostructures were used to investigate direct electron transfer (DET) of TetX2 monooxygenase (TetX2), and an enzyme-based biosensor for sensitive determination of tetracycline (TC) also fabricated. A polyethyleneimine (PEI) with positive charge groups was used for immobilization of TetX2 on modified glassy carbon electrodes. Cyclic voltammetry (CV) was employed to study the electrochemical characteristics of the immobilized enzyme and the performance of the proposed biosensor. Amongst multiple carbon-modified electrodes, nano-porous glassy carbon electrode (NPGCE) was selected because of its amplified signal response for flavin adenine dinucleotide (FAD) and superior electrocatalytic behavior toward oxygen reduction. The cyclic voltammogram of PEI/TetX2/NPGCE showed two couple of well-defined and quasi-reversible redox peaks of FAD, consistent with the realization of DET. The prepared electrode was then successfully introduced as a biosensing interface based on the oxygen reduction peak current, resulting in a linear range response from 0.5 to 5 µM with a good detection limit of 18 nM. The as-fabricated electrode demonstrates a fast response and excellent stability for the detection of TC. The results indicate that this simple, rapid, eco-friendly and economic strategy of PEI/TetX2/NPGCE preparation has potential for the fabrication of an enzyme-based biosensor for the practical detection of TC in food products.


Assuntos
Técnicas Biossensoriais , Carbono , Resíduos de Drogas/análise , Técnicas Eletroquímicas/instrumentação , Eletrodos , Enzimas Imobilizadas/metabolismo , Oxigenases de Função Mista/metabolismo , Tetraciclina/análise , Catálise , Flavina-Adenina Dinucleotídeo/análise , Oxirredução , Polietilenoimina/química , Reprodutibilidade dos Testes
13.
Pestic Biochem Physiol ; 156: 29-35, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-31027578

RESUMO

Phenacoccus solenopsis is an economically important insect pest of different agronomic and horticultural field crops. In Pakistan, the cotton crop was severely attacked by P. solenopsis during 2007 and since then a varied group of insecticides are used by farmers to manage this pest. As a result, insecticide resistance has become a barrier in control of P. solenopsis. The current study was designed to explore the basics of genetics, realized heritability and possible genetic mechanisms of resistance against spirotetramat in P. solenopsis. Before selection, the wild population (Wild-Pop) showed 5.97-fold resistance when compared with lab-reared susceptible strain (Susceptible Lab-Pop). The P. solenopsis was selected with spirotetramat to 21 generations, called Spiro-SEL Pop, which showed 463.21-fold resistance as compared with the Susceptible Lab-Pop. The values of LC50 for F1 (Spiro-SEL Pop ♂ × Susceptible Lab-Pop ♀) and F1 (Spiro-SEL Pop ♀ × Susceptible Lab-Pop ♂) populations were statistically similar and values of dominance level were 0.42 and 0.54, respectively. Reciprocal crosses between Susceptible Lab-Pop and Spiro-SEL Pop showed that resistance was of autosomal in nature with incomplete dominant traits. According to the fit test, monogenic model estimation of the number of genes, which are responsible for the development of spirotetramat resistance in a population of P. solenopsis, showed that multiple genes are involved in controlling the resistance levels in tested strains of P. solenopsis. The value of heritability for resistance against spirotetramat was 0.13 in P. solenopsis. Our results suggested the presence of a metabolic-based resistance mechanism associated with the monooxygenases in P. solenopsis, while testing the synergism mechanism. These results will provide the baseline to design an effective control strategy to manage P. solenopsis in the field.


Assuntos
Compostos Aza/farmacologia , Hemípteros/efeitos dos fármacos , Inseticidas/farmacologia , Compostos de Espiro/farmacologia , Animais , Bioensaio , Hemípteros/enzimologia , Hemípteros/metabolismo , Resistência a Inseticidas , Oxigenases de Função Mista/genética , Oxigenases de Função Mista/metabolismo , Paquistão
14.
Sci Total Environ ; 671: 696-704, 2019 Jun 25.
Artigo em Inglês | MEDLINE | ID: mdl-30939322

RESUMO

Aim of present work was to assess in-planta association potential of isolated endophytic bacterial strain Pseudomonas sp. (J10) (KY608252) with two cultivars of Lolium perenne L. (small & jumbo) and Arabidopsis thaliana L. for total petroleum hydrocarbon (TPH) degradation, alkane monooxygenase (alkb) gene expression and phytotoxicity analysis. A plant-microbe phytoremediation system was established to investigate the bacteria's ability to colonize the plant body and quantification of alkb gene to help withstand TPH stress in soil as well as in hydroponics. A real-time PCR method was developed to analyze bacterial colonization and survival within the plant body. Analysis revealed that J10 efficiently colonized all the tested plant species and expressed alkb gene under hydrocarbon stress ranging between 3.7 × 102-3.9 × 106 in A. thaliana and L. perenne (small), respectively. The colonization was more pronounced in soil as compared to hydroponic system. J10 inoculation reduced phytotoxicity and suggested that inoculation had a positive effect on plant growth under stress conditions as compared to control. L. perenne (small) showed significant TPH removal efficiency (45.6%) followed by L. perenne jumbo (24.5%) and A. thaliana (6.2%). In hydroponics, L. perenne (small) degraded about 28.2% TPH followed by L. perenne (jumbo) as 24.4%. Potential of the indigenously isolated plant endophytes may be exploited further for phytoremediation efficiency and industrial applications.


Assuntos
Biodegradação Ambiental , Lolium/microbiologia , Petróleo/metabolismo , Poluentes do Solo/metabolismo , Hidrocarbonetos/metabolismo , Oxigenases de Função Mista/genética , Oxigenases de Função Mista/metabolismo , Desenvolvimento Vegetal , Pseudomonas/fisiologia
15.
Mol Biol Rep ; 46(2): 2363-2370, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30847851

RESUMO

The gene lpmo1 encoding Penicillium verruculosum lytic polysaccharide monooxygenase (PvLPMO9A) was sequenced and homologously overexpressed in P. verruculosum B1-537 (ΔniaD) auxotrophic strain under the control of the cbh1 gene promoter in combination with either the cbh1 signal sequence (sCBH1-X series of samples) or the native lpmo1 signal sequence (sLPMO1-X series). Three enzyme samples of the sCBH1-X series were characterized by a lower overall content of cellobiohydrolases (CBHs: 26-45%) but slightly higher content of endoglucanases (EGs: 17-23%) relative to the reference B1-537 preparation (60% of CBHs and 14% of EGs), while the PvLPMO9A content in them made up 9-21% of the total secreted protein. The PvLPMO9A content in four enzyme preparations of the sLPMO1-X series was much higher (30-57%), however the portion of CBHs in most of them (except for sLPMO1-8) decreased even to a greater extent (to 21-42%) than in the samples of the sCBH1-X series. Two enzyme preparations (sCBH1-8 and sLPMO1-8), in which the content of cellulases was substantially retained and the portion of PvLPMO9A was 9-30%, demonstrated the increased yields of reducing sugars in 48-h saccharification of Avicel and milled aspen wood: 19-31 and 11-26%, respectively, compared to the reference cellulase cocktail.


Assuntos
Oxigenases de Função Mista/metabolismo , Penicillium/metabolismo , Celulase/biossíntese , Celulase/metabolismo , Celulases/genética , Celulose/genética , Celulose/metabolismo , Celulose 1,4-beta-Celobiosidase/biossíntese , Celulose 1,4-beta-Celobiosidase/metabolismo , Hidrólise , Oxigenases de Função Mista/genética , Oxigenases de Função Mista/fisiologia , Polissacarídeos
16.
Cell Mol Life Sci ; 76(12): 2329-2348, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30879092

RESUMO

Many secreted peptides used for cell-cell communication require conversion of a C-terminal glycine to an amide for bioactivity. This reaction is catalyzed only by the integral membrane protein peptidylglycine α-amidating monooxygenase (PAM). PAM has been highly conserved and is found throughout the metazoa; PAM-like sequences are also present in choanoflagellates, filastereans, unicellular and colonial chlorophyte green algae, dinoflagellates and haptophytes. Recent studies have revealed that in addition to playing a key role in peptidergic signaling, PAM also regulates ciliogenesis in vertebrates, planaria and chlorophyte algae, and is required for the stability of actin-based microvilli. Here we briefly introduce the basic principles involved in ciliogenesis, the sequential reactions catalyzed by PAM and the trafficking of PAM through the secretory and endocytic pathways. We then discuss the multi-faceted roles this enzyme plays in the formation and maintenance of cytoskeleton-based cellular protrusions and propose models for how PAM protein and amidating activity might contribute to ciliogenesis. Finally, we consider why some ciliated organisms lack PAM, and discuss the potential ramifications of ciliary localized PAM for the endocrine features commonly observed in patients with ciliopathies.


Assuntos
Chlamydomonas/enzimologia , Cílios/metabolismo , Oxigenases de Função Mista/metabolismo , Complexos Multienzimáticos/metabolismo , Peptídeos/metabolismo , Proteínas de Plantas/metabolismo , Actinas/metabolismo , Chlamydomonas/citologia , Chlamydomonas/metabolismo , Chlamydomonas/ultraestrutura , Cílios/ultraestrutura , Oxigenases de Função Mista/análise , Modelos Moleculares , Complexos Multienzimáticos/análise , Proteínas de Plantas/análise , Biossíntese de Proteínas , Transporte Proteico , Transdução de Sinais
17.
Org Lett ; 21(7): 2330-2334, 2019 04 05.
Artigo em Inglês | MEDLINE | ID: mdl-30900461

RESUMO

Ascochlorin is a medicinally important fungal meroterpenoid. Its biosynthetic pathway in Fusarium sp. was identified, and the stereoselective epoxidation of the farnesyl group by the multidomain, soluble P450 monooxygenase AscE and the subsequent formation of the unique timethylcyclohexanone ring by the membrane-bound cyclase AscF were investigated. Precursor-directed biosynthesis generated novel bromo-substituted derivatives, which exhibited potent cytotoxic activities. This study paves the way for the future metabolic engineering of medicinally important meroterpenoids for drug discovery.


Assuntos
Alcenos/química , Fungos/metabolismo , Fusarium/química , Oxigenases de Função Mista/metabolismo , Oxirredutases/metabolismo , Fenóis/química , Terpenos/química , Vias Biossintéticas , Oxigenases de Função Mista/química , Estrutura Molecular , Oxirredutases/química , Oxirredutases/isolamento & purificação , Terpenos/isolamento & purificação
18.
Biomed Pharmacother ; 113: 108712, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30849641

RESUMO

Progestins has been used widely for endometrial cancer (EC) patients. However, long term use of high dose progestin often lead to progestin resistance. Our previous studies have demonstrated that metformin reversed progestin resistance through the downregulation of the expression of glyoxalase I (GLOI) in type I endometrial cancer. Recent studies have demonstrated the role of Ten-eleven translocation 1 (TET1) in endometrial cancer, but the physiological role of TET1 in GLOI-mediated progestin resistance has been poorly addressed. Immunohistochemistry was used to detect the expression of TET1, GLOI and 5hmC in various endometrium. Western blot was carried out to analyses TET1 and GLOI expression with different treatment. Cell counting kit-8 was used to evaluate cell proliferation after various treatment. Dot blot assay and HMeDIP assay were performed to detect global hydroxmethylation levels and hydroxymethylation levels in GLOI gene respectively. In current study, we found that metformin effectively sensitized progestin in endometrial cancer cell lines through the down regulation of the expression of TET1 and GLOI. Interestingly, the exogenous increase of TET1 expression enhanced total 5hmC level and hydroxymethylation modification in glyoxalase I promoter region. This effect was abated by metformin treatment. Moreover, the expression profile of TET1 and glyoxalase I in various endometrial tissue parallelized with 5hmC level. Therefore, this finding suggests that metformin sensitized progestin in endometrial cancer through the TET1-5hmC-GLOI signaling pathway.


Assuntos
Adenocarcinoma/metabolismo , Proliferação de Células/efeitos dos fármacos , Neoplasias do Endométrio/metabolismo , Lactoilglutationa Liase/metabolismo , Metformina/farmacologia , Oxigenases de Função Mista/metabolismo , Progestinas/farmacologia , Proteínas Proto-Oncogênicas/metabolismo , Adenocarcinoma/patologia , Linhagem Celular Tumoral , Regulação para Baixo , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Sinergismo Farmacológico , Neoplasias do Endométrio/patologia , Feminino , Expressão Gênica/efeitos dos fármacos , Humanos , Lactoilglutationa Liase/genética , Metformina/administração & dosagem , Oxigenases de Função Mista/genética , Progestinas/administração & dosagem , Proteínas Proto-Oncogênicas/genética
19.
Nat Commun ; 10(1): 960, 2019 02 27.
Artigo em Inglês | MEDLINE | ID: mdl-30814511

RESUMO

Genetic diversity is a result of evolution, enabling multiple ways for one particular physiological activity. Here, we introduce this strategy into bioengineering. We design two hydroxytyrosol biosynthetic pathways using tyrosine as substrate. We show that the synthetic capacity is significantly improved when two pathways work simultaneously comparing to each individual pathway. Next, we engineer flavin-dependent monooxygenase HpaBC for tyrosol hydroxylase, tyramine hydroxylase, and promiscuous hydroxylase active on both tyrosol and tyramine using directed divergent evolution strategy. Then, the mutant HpaBCs are employed to catalyze two missing steps in the hydroxytyrosol biosynthetic pathways designed above. Our results demonstrate that the promiscuous tyrosol/tyramine hydroxylase can minimize the cell metabolic burden induced by protein overexpression and allow the biosynthetic carbon flow to be divided between two pathways. Thus, the efficiency of the hydroxytyrosol biosynthesis is significantly improved by rearranging the metabolic flux among multiple pathways.


Assuntos
Oxigenases de Função Mista/metabolismo , Álcool Feniletílico/análogos & derivados , Vias Biossintéticas , Evolução Molecular Direcionada , Escherichia coli/genética , Escherichia coli/metabolismo , Engenharia Metabólica/métodos , Oxigenases de Função Mista/genética , Modelos Biológicos , Álcool Feniletílico/metabolismo , Especificidade por Substrato
20.
Food Chem ; 288: 368-376, 2019 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-30902306

RESUMO

Exogenous Ca2+ affects the phenolic metabolism and physiological indices of germinated wheat under ultraviolet-B (UV-B) radiation, but the mechanism is unclear. The current study applied exogenous Ca2+ and Ca2+ channel blocker LaCl3 to the germinated wheat under UV-B radiation to unravel the role of Ca2+. The results indicated that total phenolic content (TPC) of the 4-day germinated wheat under UV-B radiation with CaCl2 (UV-B+Ca) treatment significantly increased by 10.3% as compared to the UV-B treatment. Gene expression levels of p-coumarate 3-hydroxylase, cinnamic acid 4-hydroxylase and caffeic acid O-methyltransferase were positively correlated with the content of ferulic and p-coumaric acids, respectively. Exogenous Ca2+ could significantly alleviate the membrane lipid peroxidation, activate the antioxidant enzymes and regulate the phytohormone level under UV-B radiation. This study suggested that exogenous Ca2+ participated in the phenolic metabolism and physiological regulation in germinated wheat under UV-B radiation.


Assuntos
Germinação/efeitos da radiação , Fenóis/metabolismo , Triticum/crescimento & desenvolvimento , Raios Ultravioleta , Antioxidantes/metabolismo , Cloreto de Cálcio/química , Cloreto de Cálcio/farmacologia , Cromatografia Líquida de Alta Pressão , Expressão Gênica/efeitos dos fármacos , Expressão Gênica/efeitos da radiação , Germinação/efeitos dos fármacos , Malondialdeído/análise , Metiltransferases/genética , Metiltransferases/metabolismo , Oxigenases de Função Mista/genética , Oxigenases de Função Mista/metabolismo , Fenóis/análise , Reguladores de Crescimento de Planta/análise , Triticum/química , Triticum/metabolismo
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